Simultaneously HPLC Analysis of B1, B9 and B12 Vitamins at Trace Levels via Cloud Point Extraction

H. I. Ulusoy, İpek Nur Yi̇ği̇t, Ümmügülsüm Polat, Esra Durgun, Aslıhan Gürbüzer, Songül Ulusoy
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Abstract

An enrichment and determination method based on liquid chromatographically analysis and cloud point extraction (CPE) has been developed for trace levels of B vitamins (B1, B9 and B12) in the proposed study. Vitamin molecules were drawn into the non-ionic surfactant phase of Polyethylene Glycol (PEG-6000) in the presence of pH 9.0 medium. The surfactant-rich phase separated by centrifugation and then dissolved with 700 µL of ethanol. The obtained ethanol phase was filtered by 0.45-micron filter prior to the HPLC analysis. All parameters affecting the CPE method such as pH, buffer volume, incubation time, surfactant and electrolyte concentration, solvent for the surfactant-rich phase and its amount have been individually studied and optimized step by step. After the optimization of all parameters of the CPE process, the detection limits of the developed method for B1, B9 and B12 vitamins were calculated as 1.42 ng mL-1, 7.14 ng mL-1 and 14.28 ng mL-1 , respectively. The linear working ranges for three vitamin molecules was obtained in the range of 5.0-500.0 ng mL-1. After CPE procedure, determination of vitamin molecules was carried out by using HPLC system with diode array detector(DAD) at 244 nm for vitamin B1, 285 nm for vitamin B9, and 361 nm for vitamin B12, respectively
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通过云点萃取同时对痕量水平的 B1、B9 和 B12 维生素进行高效液相色谱分析
在拟议的研究中,针对痕量 B 族维生素(B1、B9 和 B12),开发了一种基于液相色谱分析和浊点萃取(CPE)的富集和测定方法。在 pH 值为 9.0 的介质中,维生素分子被萃取到聚乙二醇(PEG-6000)的非离子表面活性剂相中。富含表面活性剂的相通过离心分离,然后用 700 µL 乙醇溶解。得到的乙醇相在进行 HPLC 分析前用 0.45 微米过滤器过滤。对影响 CPE 方法的所有参数,如 pH 值、缓冲液量、孵育时间、表面活性剂和电解质浓度、富含表面活性剂相的溶剂及其用量,都进行了单独研究和逐步优化。在对 CPE 过程的所有参数进行优化后,计算得出所开发方法对 B1、B9 和 B12 维生素的检出限分别为 1.42 ng mL-1、7.14 ng mL-1 和 14.28 ng mL-1。三种维生素分子的线性工作范围为 5.0-500.0 ng mL-1。经过 CPE 程序后,使用带有二极管阵列检测器(DAD)的 HPLC 系统对维生素分子进行测定,维生素 B1 的检测波长为 244 nm,维生素 B9 的检测波长为 285 nm,维生素 B12 的检测波长为 361 nm。
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